Clonal expansion of T cells in response to cognate antigen eventually leads to deletion of most of the expanded T cells via programmed cell death (apoptosis), with only a minority surviving to become memory cells. This process is crucial for restoring T cell homeostasis after termination of an acute immune response, which promotes protective immunity and deters autoimmunity. While there is now strong evidence that proteins of the Bcl-2 family, particularly anti-apoptotic Bcl-2 and pro-apoptotic Bim, play an important role in mediating apoptosis of T cells activated in vivo, little is known about how these proteins themselves are controlled. One mechanism by which Bcl-2 family members may be regulated is ubiquitin/proteasome-mediated degradation. T cells have specialized proteasomes called "immunoproteasomes" that contain three interferon-g-inducible catalytic immunosubunits: LMP2, LMP7 and MECL-1. We have recently observed that T cells from immunoproteasome-deficient mice lacking both MECL-1 and LMP7 exhibit exaggerated apoptosis after in vivo activation by super-antigen. Conversely, over-expression of LMP7 in normal super-antigen activated T cells significantly suppresses apoptosis. Taken together, these results suggest that immunoproteasomes play a role in regulating the magnitude of apoptosis of activated T cells. Given the central role of Bcl-2 family members in mediating activated T cell apoptosis, and that several Bcl-2 family members are proteasome substrates, we hypothesize that immunoproteasomes regulate the magnitude of Bim-driven apoptosis of activated T cells through degradation of Bcl-2 family members. We will investigate this hypothesis in the following specific aims: 1. Determine the role of immunoproteasomes in the degradation and function of pro-apoptotic Bcl-2 family members. This aim will involve the generation of triple knockout mice (Bim-/-/MECL- 1-/-/LMP7-/-) to determine if Bim is required for the pro-apoptotic effect of immunoproteasome deficiency, and this aim will also assess the impact of immunoproteasome deficiency on the function and degradation of pro- apoptotic Bcl-2 family members in activated T cells. 2. Identify structural and functional features of immunoproteasomes that are critical for their anti-apoptotic activity. This aim will involve testing the ability of active site and assembly mutants of LMP7 and related proteasome subunits to modulate apoptosis of activated T cells, and determine whether immunoproteasome expression in normal activated T cells correlates with apoptotic activity. In the long-term, elucidation of molecular mechanisms underlying the role of immunoproteasomes in activated T cell apoptosis will further our understanding of the development of immune memory, and potentially identify targets for enhancing apoptosis of auto-reactive T cells involved in the pathogenesis of autoimmune disease, or suppressing apoptosis of T cells that could contribute to control of infectious or malignant disease. An immune response to a "threat" (such as infection or tumor) must be controlled in such a way as to provide memory to protect from similar threats in the future (immunity), while at the same time preventing unwanted damage to one's self (autoimmunity). The goal of this project is to improve our understanding of the molecules that participate in this control system, which could lead to better treatments of diseases in which this system is self-defeating or breaks down (infections, cancer, or autoimmune disease). [unreadable] [unreadable] [unreadable]